ffucal I '6Manp l-4glcnacp1-46lcnac 15.1% Galpl-4GlcNA~f31-2Manal~~ f[f(glcnacp-)manl

Transcription

1 Eur. J. Biochem. 192, (1990) 0 FEBS 1990 The spectrum of N-linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells Chun-Ting YUEN', Steven A. CARR' and Ten FEZ' ' Glycoconjugates Section, Medical Research Council Clinical Research Centre, Harrow, England ' Department of Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, USA (Received April 26, 1990) - EJB Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (scd4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S. A., Hemling, M. E., Folena-Wasserman, G., Sweet, R. W., Anumula, K., Barr, J. R., Huddleston, M. J. & Taylor, P. (1989) J. Bid. Chem. 264, Among oligosaccharides released from scd4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of scd4 as follows : ffucal Gal@l-4GlcNAcpl-2Manal '6Manp l-4glcnacp1-46lcnac 15.1% Galpl-4GlcNA~f31-2Manal~~ f[f(glcnacp-)manl ' ~FUC MandlcNAc4lcNAc 5.3% Gal@---GlcNAc@-Man \.Man-cNAc----GlcNAc Galp41cNAcpl.4 2Man / Gal@41cNAc@ld ffuc 0.7% 0.9% where t- indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4. The CD4 glycoprotein of human T-lymphocyte membranes, a member of the immunoglobulin superfamily and an important regulator of T-lymphocyte responses [l, 21 is also Correspondence to T. Feizi, Medical Research Council Clinical Research Centre, Watford Road, Harrow HA1 3UJ, England Abbreviations. ConA, concanavalin A; E-PHA, Phaseolus vulxaris erythrohaemagglutinin; Glc-olNAc, N-acetylglucosaminitol; HV-1, human immunodeficiency virus 1 ; gp120, envelope glycoprotein of molecular mass 120 kda of HV-1; RCA-120, Ricinus communis agglutinin 120; scd4, soluble recombinant CD4 glycoprotein derived from Chinese hamster ovary cells. Enzymes. /?-N-Acetylhexosaminidase (EC ); b-galactosidase (EC ); a-mannosidase (EC ); 8-mannosidase (EC ); sialidase (EC ). the major cellular receptor for the human immunodeficiency virus 1 (HV-1) [3-51. Observations on the high affinity of interaction of CD4 glycoprotein with the envelope glycoprotein of molecular mass 120 kda (gp120) of HV-1 [6] have led to the generation of recombinant soluble forms of CD4 lacking the membrane-spanning and cytoplasmic domains for use as inhibitors of HV-1 attachment and infectivity [ One such recombinant CD4 produced in Chinese hamster ovary cells (scd4) and shown to be a powerful inhibitor of HV-1 infection of CD4' cells has been investigated recently by mass spectrometry [12], and it has been established that both protein N-glycosylation sites (Asn271 and Asn300) have oligosaccharides attached, with the majority of components at each site being complex type biantennary sialo-oligosaccha-

2 524 rides. We report here results of enzymic microsequencing of the N-linked oligosaccharides of scd4 following their release from the protein by hydrazinolysis. EXPERMENTAL PROCEDURES CD4 glycoprotein The recombinant, human scd4 glycoprotein expressed in Chinese hamster ovary cells [7] was purified to apparent homogeneity from serum-free conditioned medium, and glycopeptides containing either Asn271 or Asn300 were prepared as described elsewhere [12]. Chemicals and enzymes NaB3H4 (5 Ci/mmol) was purchased from Amersham nternational (Bucks, England). The following glycosidases were from Seikagaku Kogyo Co. (Tokyo, Japan) : P-galactosidase, P-N-acetylhexosaminidase and a-mdnnosidase purified from jack bean meal. The latter was found to contain, in addition, P-mannosidase activity when tested with a standard oligosaccharide Man3-GlcNAc-[3H]Glc-olNAc (Glc-olNAc, N-acetylglucosaminitol) derived from human transferrin oligosaccharides. Aspergillus saitoi a-mannosidase which specifically cleaves Manal-2Man linkages [31 purified from Morushin [14,15] was a gift from Dr T. Mizuochi (Fujita-Gakuen Health University School of Medicine, Toyoake Aichi, Japan). Sialidase purified from Arthrobacter ureafaciens was purchased from Nakarai Chemicals Ltd (Kyoto, Japan). Digestions of radioactive oligosaccharides with glycosidases were performed as described previously [13,16]. Affinity columns of Ricinus communis agglutinin 120 (RCA-120) and of Phaseolus vulgaris erythrohaemagglutinin (E-PHA) coupled to agarose were from Vector Laboratories (Peterborough, UK) and concanavalin A (ConA) coupled to Sepharose from Pharmacia (Uppsala, Sweden). Oligosaccharides from CD4 glycoprotein A sample (1.5 mg) of scd4 glycoprotein was subjected to hydrazinolysis and the oligosaccharides were separated from peptides by paper chromatography essentially as described in [17]. The oligosaccharide fraction was reduced in the presence of 1.2 pmol NaB3H4, followed by 33 pmol NaBH, (yield, 3.9 x 10 cpm). Analytical methods High-voltage paper electrophoresis, Bio-Gel P-4 (extrafine) column chromatography and enzymic microsequencing procedures were performed according to procedures described previously [16, 18, 191. Affinity chromatography Exploratory experiments showed that sialidase-treated oligosaccharides from scd4 glycoprotein were in part retarded or retained by affinity columns of RCA-120 and ConA but not E-PHA. The last observation indicated that certain galactose-terminating biantennary and triantennary complextype oligosaccharides [20] with bisecting N-acetylglucosamine residues were not present. The following fractionation protocol was adapted from published procedures [ Sialidase-treated radioactive oligosaccharides from scd4 glyco Fraction Number Fig. 1. Separation of radiolabelled sialidase-treated oligosaccharides on an affinity column of RCA-120. Radioactive oligosaccharides from scd4 glycoprotein were treated with sialidase and chromatographed on a RCA-120 lectin affinity column (0.5 cm x 48 cm; flow rate, 6 ml/h). The radioactivity in each tube (1.25 ml/tube) was determined by liquid scintillation counting. The unretained oligosaccharides (), the retarded oligosaccharides (1 and 111) and the oligosaccharides eluted in the presence of 100 mm lactose (V) were pooled as indicated by the bars. Arrow indicates point of application of 100 mm lactose protein (4 x lo6 cpm) were chromatographed using a RCA- 120 affinity column (0.5 cm x 48 cm) equilibrated in 10 mm Tris/HCl buffer, ph 7.5, containing 0.1 M NaCl (flow rate, 6.0 ml/h; fraction size, 1.25 ml). An excluded fraction,, and two retarded fractions, 1 and 111, were obtained. A fourth fraction, V, was eluted in the presence of 100 mm lactose in the same buffer (Fig. 1). The pooled fractions -V were desalted with Bio-Rad AG SOW-X 12 (H form) and Bio-Rad AG 3-X 4 (OH- form). Fraction V was further chromatographed using a Bio-Gel P-2 column (1.5 cm x 100 cm) in order to remove lactose. Fractions -1V (10-50 x lo4 cpm of each) were then chromatographed on a ConA affinity column (0.9 cm x 3 cm) equilibrated in 10 mm Tris/HCl buffer, ph 7.5, 0.1 M NaCl (flow rate, 12 ml/h; fraction size, 1.2 ml). n each case 18 fractions were collected in equilibration buffer, followed by 18 fractions in the presence of 20 mm methyl-a-d-glucopyranoside in the same buffer, and finally 10 fractions in the presence of 500 mm methyl-a+mannopyranoside. A total of 10 pooled fractions were thus obtained (Fig. 2), designated as E (excluded), R (retarded), G (eluted in the presence of methyl-a-d-glucopyranoside) and M (eluted in the presence of methyl-sr-d-mannopyranoside). These were desalted and freed of methyl-a-d-glycopyranosides by ion-exchange and Bio-Gel P-2 chromatography respectively as described above. n six of the fractions, - M, 11-E, - G, 111-G, V-E and V-G, several distinct oligosaccharide peaks chromatographing between 17 and 9 glucose units on a Bio-Gel P-4 column, and accounting for over 80% of total radioactive counts, were subjected to enzymic microsequencing. The remaining radioactive counts chromatographed as small peaks between 21 and 11 glucose units but were not investigated further. RESULTS AND DSCUSSON When the radiolabelled oligosaccharides of scd4 were subjected to paper electrophoresis at ph 5.4, a minor neutral fraction (1 2.8% calculated from the radioactive counts) and two acidic fractions A1 (57.7%) and A2 (29.5%) were obtained (Fig. 3 A). Upon digestion with sialidase, the acidic fractions were completely converted into neutral components

3 Con A Sialidase Treated Oligosaccharides 1 ll N Con A 1 ConA Con A E R G M E R G G E G Fig. 2. Summary of the fractionation of sialidase-treated total oligosaccharide mixture obtained from scd4 glycoprotein by sequential chromatographies on an affinity column of RCA-20 and ConA as described in Experimental Procedures. The numbers under each fraction refer to the yields as the molar percentage of the total oligosaccharides (based on radioactivities). Designation of fractions: E and R, excluded and retarded by the ConA column, respectively; G and M, eluted from the ConA column in the presence of methyl-a-d-glucopyranoside and methyl-a-d-mannopyranoside, respectively Distance from origin lcrnl Fig. 3. High-voltage paper electrophoresis qf radioactive oligosaccharides released from scd4 glycoprotein. Total radioactive oligosaccharides obtained from scd4 were subjected to high-voltage electrophoresis at ph 5.4 before (A) and after (B) treatment with sialidase. Arrows 1 and 2 indicate positions of lactitol and sialyllactitol standards, respectively. Areas shown by bars as Mono and Di are, respectively, the positions of migration of monosialykdted and disialylated oligosaccharides from human gg. The radioactive profiles have been normalised so that the heights of the maximum peaks in the two panels are comparable (Fig. 3B) indicating that the acidic groups were due to the presence of sialic acid residues. Fractions A1 and A2 electrophoresed in positions identical to monosialylated and disialylated biantennary complex-type oligosaccharides, respectively, obtained from human gg (Fig. 3A). n earlier methylation analyses of oligosaccharides released from scd4 glycopeptides [ 121 using peptide N-glycanase, terminal and C3-substituted galactose residues were detected at a ratio of 1 : 2, and C6-substituted galactose residues were not detected. From these results, and the results of enzymic microsequencing (see below), it is deduced that fractions A1 and A2 consist predominantly of monosialylated and disialylated biantennary oligosaccharides of complex type with sialic acid residues linked to the C3 position of sub-terminal galactose residues. Oligosaccharide structures identified after desialylation Oligosaccharide structures in major fraction 111-G andminor fraction V-G. The oligosaccharides in the major fraction 111-G (75.1% of total radioactive oligosaccharides) and the minor fraction V-G (2.2%) which were retarded and retained respectively by the 1-galactoside-specific RCA-120 affinity column, were bound by the ConA affinity column and were eluted in the presence of 20 mm methyl a-d-glucopyranoside (Fig. 2). The oligosaccharides in fraction 111-G and approximately one quarter of the oligosaccharides in fraction V-G chromatographed on the Bio-Gel P-4 column as doublets at about 14 and 13 glucose units (results for 111-G are shown in Fig. 4A) the peak at about 13 glucose units being at the position of elution of asialo-digalactosyl biantennary complex-type oligosaccharide from human transferrin: Galfil-4GlcNAcfl1-2Manal-6(Gal~1-4GlcNAc~1-2Manal-3)Man~1-4GlcNAc B1-4[3H]Glc-olNAc. The peak shifts after treatment with jack bean p-galactosidase followed by jack bean p-n-acetylhexosaminidase (loss of 2 glucose units followed by loss of 4 glucose units) corresponded to the release of two galactose residues followed by two N-acetylglucosamine residues. n the case of fraction 111-G, further digestion with jack bean a- plus P-mannosidases gave two radioactive peaks chromatographing at the positions of standard oligosaccharides GlcNAc- (F~c)[~H]Glc-olNAc and GCNAC[~H]G~C-~~NAC consistent with the release of three core-region mannose residues. n order to ensure that the relative positions of the two components were not reversed following treatments with p- galactosidase and j-n-acetylhexosaminidase, additional digestion experiments were performed with the two peaks (1 and 2) after they were separately pooled as indicated by the bars in Fig. 4A. The relative positions of the products (indicated by the large arrowheads) were maintained. From these results and methylation analyses performed earlier [ 121 which have shown the presence of CCsubstituted and C4,6-substituted N-acetylglucosamine, C2-substituted and C3,6-substituted mannose residues, as well as terminal fucose residues, these oligosaccharides were identified as a mixture of the digalactosyl biantennary complex-type chains with or without core-region fucose residues as shown in Fig. 5. The presence of the digalactosyl biantennary oligosaccharides in fraction V-G can be explained by our observations (results not shown) that, depending on the amounts of radioactive oligosaccharides applied, all or a part of these oligosaccharides are retained by this batch of RCA-120 agarose affinity column under the chromatography conditions used here. These can be specifically eluted in the presence of lactose. Oligosaccharide structures ident fied in minoryraction 11-G. The oligosaccharides in this fraction (12.6% of total radioactive oligosaccharides) were retarded by the RCA-120 affinity column (Fig. 1) and were bound by the ConA affinity column and were eluted in the presence of 20 mm methyl a-dglucopyranoside (Fig. 2). Approximately one sixth of the oligosaccharides in this fraction (2.1% of total radioactive oligosaccharides), designated 11-Ga, eluted on the Bio-Gel

4 526 Glucose Units C-G/V-G 39.0% 36.7% 11-G(a) Fuc Man4lcNAc-lcNAcOT GalB+lcNAcB-Man/ 1.3% / Man--GlcNAc41cNAcoT 0.8% > L >._._ L u m m a 11-G(b) 11-E GalB+lcNAc@-Man/ GalB+lcNAcB-Man/ GalB---GlcNAc$-Man Fuc Man Man--GlcNAc-lcNAcoT Man Man41cNAc41cNAcoT Fuc /MandlcNAc41cNAcoT 1.3% 0.9% 0.6% Gal@+lcNAcB-Man MandlcNAc--G1cNAcoT 0.4% V-E FUC Man4lcNAc4lcNAcOT GalB----GlcNAcBl. 4Man / Gal@--GlcNAc B % Retention Time lhl Fig. 4. Bio-Gel P-4 chromatography profiles of oligosaccharide peaks subjected to enzymic microseyuencing. Unbroken lines in (A- D) are the radioactive peaks in fractions 111-G, 11-C, 11-E and V-E, respectively, before jack-bean 8-gakactosidase treatment, and in (E) are the peaks in fraction - M before A. saitoi al-2mannosidase treatment. ) Profiles after the respective treatments with p-galactosidase (A-D) and xl-2mannosidase (E). (----) Profiles after treatment with jack bean fl-n-acetylhexosaminidase. (-.-.-) Profiles after treatment with jack-bean G- plus p-mannosidases. n (A) horizontal bars designated 1 and 2 indicate the radioactive fractions pooled in the second set of enzymatic digestion experiments performed with 111-G, as described in Results and Discussion; the large arrowheads 1 and 2 indicate the positions of the corresponding products obtained after the digestion with /-galactosidase followed by P-N-acetylhexosaminidase. n (B) the letters a and b refer to radioactive peaks given by fractions T-Ga and 11-Gb respectively; ajb indicates that a similar radioactive profilc was obtained with fractions T-Ga and b, and the results shown are for 11-Gb. The positions of elution of the following standard oligosaccharides are shown: S1, digalactosyl biantennary complex-type asialo-oligosaccharide Galbl-4GlcNAcp-2Manal-6- (Gal,81-4GlcNAc~l-2Man~11-3)Man~l-4GlcNAc~3-4[3H]Glc-olNAc derivcd from human transferrin after treatment with sialidase; S2 and S3. core region structures Man,-GlcNAc(F~c)[~H]Glc-olNAc and Ma~i~-GlcNAc[~H]Glc-olNAc, respectively, derived from human gg oligosaccharides after sequential treatments with sialidase, P-galactosidase and P-N-acetylhexosaminidase; S4 and SS, core-region structures GlcNAc-(F~c)[~H]Glc-olNAc and GlcNAc[3H]Glc-olNAc derived from human gg and human transferrin oligosaccharides, respectively, after sequential treatments with sialidase, and jack-bean p- galactosidase, P-N-acetylhexosaminidase and G- plus P-mannosidases. The radioactive profiles are normalised so that the height of the maximum peak in each panel is the same or nearly the same -M GalB*lcNAc@-Man GalB---GlcNAcB1.4 GalB---GlcNAcBl MandlcNAc41cNAcoT / 2Man Man Man /Man Man--GlcNAc--G1cNAcoT Man r- Man. Man Manal-2 L Man Man41cNAc41cNAcOT Man (Manal-22 [ : 1: :l >Man4lcNAc4lcNAcOT (Manal-2)3 [ 11: > MandlcNAcdlcNAcOT 0.3% 0.5% 0.2% 0.1% Fig. 5. Proposed structures for oligosaccharides contained in fraction l-g, 11-G, l-e, V-E and --M derived from scd4 glycoprotein. Except in the case of the major components (111-G/V-G) the monosaccharide configurations and linkages were mostly not determined. n the case of the triantennary oligosaccharides (V-E), the presence of the alternative isomers (C2,6-substituted) in trace amounts could not be excluded. The percentages (determined on the basis of radioactivity), for individual structures identified in the various fractions, refer to the proportions of total sialidase-treated oligosaccharides. GlcNAco,, 3H-labelled N-acetylglucosaminitol P-4 column between 13 and 12 glucose units (Fig. 4B). The products after treatment with jack bean 6-galactosidase chromatographed as a doublet (at about 12 and 11 glucose units) corresponding to the release of one galactose residue. 0.1%

5 521 Further digestion with jack bean P-N-acetylhexosaminidase gave peak shifts (loss of 4 glucose units) corresponding to the release of two N-acetylglucosamine residues. The resultant radioactive products eluted at the same position as the standard trimannosyl core structures with and without core-region fucose residues, Man3-GlcNAc-(Fuc)[3H]Glc-olNAc and Man3-GlcNAC[3H]Glc-olNAc respectively. n a separate experiment where the original fraction 11-Ga was first treated with jack bean fl-n-acetylhexosaminidase, loss of one N- acetylglucosamine residue occurred (results not shown). Together, these results are consistent with the presence in - Ga of a mixture of monogalactosyl biantennary complex-type oligosaccharides with and without core-region fucose residues as shown in Fig. 5. About a further one sixth of oligosaccharides in fraction 11-G, designated 11-Gb (2.2% of total radioactive oligosaccharides) chromatographed on the Bio-Gel P-4 column between ll and 10 glucose units (Fig. 4B). The products after treatment with jack bean P-galactosidase chromatographed as a doublet (at about 10 and 9 glucose units) corresponding to the release of one galactose residue. Further digestion with jack bean fl-n-acetylhexosaminidase gave peak shifts (loss of 2 glucose units) corresponding to the release of one N- acetylglucosamine residue. The resultant radioactive products eluted at the same position as the standard trimannosyl core structures with or without core-region fucose residues as described above. On the basis of these results, the oligosaccharides in fraction 11-Gb were identified as monogalactosyl monoantennary complex-type oligosaccharides with trimannosyl cores with and without core-region fucose residues as shown in Fig. 5. Oligosaccharide structures identqied in minor jraction -E. The oligosaccharides in this fraction (1.2% of total radioactive oligosaccharides), which were retarded by the RCA-120 affinity column, were excluded by the ConA affinity column. The majority of oligosaccharides in this fraction chromatographed on the Bio-Gel P-4 column between 10 and 9 glucose units (Fig. 4C). The products after treatment with jack bean p- galactosidase chromatographed as a doublet (at about 9 and 8 glucose units) corresponding to the release of one galactose residue. Further digestion with jack bean fl-n-acetylhexosaminidase gave peak shifts (loss of 2 glucose units), corresponding to the release of one N-acetylglucosamine residue. When the radioactive products were further digested with jack bean M- plus P-mannosidases, the peak shifts corresponded to the release of two mannose residues. The residual radioactive products chromatographed at the same position as the core structures GlcNAc-( & F~c)[~H]Glc-olNAc. From these results, these oligosaccharides were identified as monogalactosyl monoantennary chains with dimannosyl cores with and without core-region fucose residues as shown in Fig. 5. Oligosaccharide structures identified in minor fraction V-E. The oligosaccharides in this fraction (1.6% of total radioactive oligosaccharides) which were retained by the RCA-120 affinity column and were eluted in the presence of 100 mm lactose, were excluded by the ConA affinity column. Approximately half of these oligosaccharides chromatographed on the Bio- Gel P-4 column between 17 and 16 glucose units (Fig. 4D). The products after treatment with jack bean fl-galactosidase chromatographed as a peak at about 14 and a shoulder at about 13 glucose units, corresponding to the release of three galactose residues. Further digestion with jack bean fl-nacetylhexosaminidase gave two peaks (loss of 6 glucose units) corresponding to the release of three N-acetylglucosamine residues. The residual radioactive products chromatographed at the same position as the standard trimannosyl core structures Man3-GlcNAc( F~c)[~H]Glc-olNAc. From these results and the methylation analyses [21 which have shown the presence of C2,Csubstituted mannose residues, these oligosaccharides were identified as triantennary complex-type structures such as those shown in Fig. 5. Oligosaccharide structures identified in minor fraction - M. n this minor fraction (1.3% of radioactive oligosaccharides) a series of high mannose-type oligosaccharides Man,GlcNAc2 (0.5%), Man,GlcNAc, (0.2%), Man,Glc- NAcz (0.1 YO) and Man,GlcNAc2 (0.1 YO) were identified from the following results. First, the radioactive oligosaccharides were not bound by the fl-galactoside-specific RCA-120 affinity column (Fig. l), but were bound by the ConA affinity column, and were eluted in the presence of 500 mm methyl-a-d-mannopyranoside (Fig. 2). Second, their elution positions on the Bio-Gel P-4 column (Fig. 4E) were at about 9, 10, 11 and 12 glucose units, respectively. Third, the elution positions were unaffected after treatment with jack bean /3-N-acetylhexosaminidase. Fourth, following incubation with A. saitoi a1-2 mannosidase, a product eluting at about 9 glucose units was obtained corresponding to the release of 0,1,2 and 3 mannose residues respectively. Fifth, subsequent digestion with jack bean M- plus p-mannosidases, gave a radioactive product eluting at 4 glucose units corresponding to G~cNAc[~H]G~colNAc. Consistent with these results was the detection of small amounts of terminal mannose residues by methylation analyses [ 121. Conclusion n this study over 80% of the radioactive oligosaccharides were identified, with biantennary complex-type structures as the major components (77.8%) and monoantennary and triantennary complex-type and high-mannose-type structures as minor components (3.2, 0.7 and 0.9%, respectively). These numbers are in good agreement with the estimates made by mass spectrometry [12]. A total of fourteen oligosaccharide structures were identified after desialylation. However the number of oligosaccharide structures is likely to be much greater, since among the total radioactive oligosaccharides before sialidase treatment approximately 12% (12.8% minus the high-mannose-type structures 0.9%) are deduced to be asialo forms and approximately 87% monosialo or disialo forms of complex-type chains. The oligosaccharide structures released by hydrazinolysis from the individual glycopeptides containing Asn271 or Am300 were not investigated in detail in the present study. However, their chromatographic profiles on Bio-Gel P-4 after sialidase treatment and sequential chromatography on the two lectin columns (results not shown) suggest that the 14 structures identified among the total scd4 oligosaccharides are similarly represented at the two glycosylation sites. This would argue against the possibility that the minor oligosaccharide structures are derived from impurities in the scd4 preparation. Nonetheless, the finding of several different variants of the major biantennary oligosaccharides indicates that alternative oligosaccharide structures occur at each glycosylation site and that multiple glycosylation variants of scd4 glycoprotein are possible. Glycosylation of CD4 protein has been shown to be necessary for its expression at the cell surface [24]. However, recombinant soluble CD4 proteins that lack both glycosylation sites bind to the HV-1 envelope glycoprotein gp120 with an affinity similar to that of the full-length receptor, and inhibit virus infection in vitro [lo, 11, 25, 261. Thus the N-linked

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